Isolation of Western Equine Encephalomyelitis (Wee) Virus From Mosquitoes In Saskatchewan, 1962

Saskatchewan, in the summer of 1962, was the scene of an extensive outbreak of western equine encephalomyelitis (WEE) in horses. The results of mosquito survey work showed Culiseta inornata and Culex tarsalis respectively to be the two most abundant mosquito species during midsummer. These species are those reported to be most commonly associated with outbreaks of WEE. Five hundred and sixty-four pools of mosquitoes were examined for the presence of WEE virus. Six pools, three of C. tarsalis and one each of C. inornata, Aedes flavescens and Aedes dorsalis, yielded WEE virus. Positive mosquitoes were from St. Walburg (C. inornata), Saskatoon (C. tarsalis — two, A. dorsalis — one), Outlook (C. tarsalis) and Kisbey (A. flavescens).     

Antibody Against Western Encephalitis Virus Occurring in the Serum of Garter Snakes (Colubridae: Thamnophis) in Saskatchewan

A study was made of feeding and temperature as factors affecting the appearance of western equine encephalitis (WEE) virus-neutralizing serum (VNS) antibodies in the serum of garter snakes (Thamnophis spp). Eighty snakes were collected in the field, held in captivity under controlled conditions, and bled at frequent intervals. The sera were examined by standard procedures for the presence of WEE VNS-antibodies. It was found that snakes held between 10-28°C showed conversion and intermittent WEE VNS-antibody appearance, whereas snakes held at 6°C showed a decline in titre. The appearance of WEE VNS-antibody was related to environmental temperature, or a temperature-controlled factor, and not to feeding.     

The Kinetics of Hematopoiesis in the Light Horse I. The Lifespan of Peripheral Blood Cells in the Normal Horse

Three Standardbred horses were given 0.2 mg (1 mCi) of ⁷⁵selenomethionine intravenously and a second group of three were given 10 mCi of tritiated diisopropylfluorophosphate (0.5 mg) intravenously. Observations on labeled cells were continued for 250 days after radioselenium injection and 160 days after tritium injection.

The lifespan of erythrocytes using ⁷⁵selenmethionine was 155 ± 10 days and 148 ± 7.8 days using tritiated diisopropylfluorophosphate. There was no significant difference at the 10% level between the lifespans, using these labels. The uptake of radioselenium into erythrocytes reached a mean maximum of 11.5% at 82 ± 18.9 days after injection of the label. There was an elution of from 17.6% of the injected dose of tritium label down to 7.5% eight days after injection of this isotope. From this study both of these labels appear to be satisfactory for determining the erythrocyte lifespan of the horse.

The mean time of the curve of mean radioselenium activity of peripheral blood leukocytes for the three horses was 5.54 days and the lifespan of these cells was seven days. The mean lifespan of peripheral blood leukocytes of the three horses after in vivo labelling with tritiated diisopropylfluorophosphate was 17.3 hours. No specific labelling was found in bone marrow or peripheral blood cells at this level of tritium labelling by dipping emulsion autoradiography.

The mean lifespan of equine platelets for three horses using radioselenomethionine was 9.2 days and the mean lifespan of equine platelets using tritiated diisopropylfluorophosphate was 6.6 days in a group of three horses.

     

Amount of western equine encephalitis virus inoculated by transmitting Culex tarsalis mosquitoes.

The amount of western equine encephalitis virus inoculated by infected Culex tarsalis mosquitoes is highly variable between individual mosquitoes. The majority (68 per cent) inoculate less than 100 intracerebral three-week-old mouse LD50 at time of feeding and the quanity appears unrelated to temperature or length of incubation following initial infection.     

Effect of storage time and temperature on the total protein concentration and electrophoretic fractions in equine serum

Serum protein electrophoresis (SPE) is a technique that could be considered one of the most useful diagnostic aids available to the clinician. The effect of storage time and temperature on the total proteins and electrophoretic fractions (albumin, α₁-, α₂-, β₁-, β₂-, and γ-globulins) was assessed in 24 healthy horses. All samples, collected by jugular vein puncture, were centrifuged and divided into 4 aliquots. The 1st aliquot was analyzed within 3 h from collection (time 0), the 2nd was refrigerated at +4°C for 24 h, the 3rd was refrigerated at +4°C for 48 h, and the last was frozen at −20°C for 48 h. One-way repeated-measures analysis of variance (ANOVA) showed a significant effect (P < 0.05) of the different storage conditions on the concentrations of all the parameters studied and significant variations in the percentages of albumin, α₁-globulins, α₂-globulins, and γ-globulins. Compared with time 0 the total protein concentration increased significantly after 48 h at −20°C, the albumin percentage decreased after 48 h at −20°C, the α₁-globulin percentage increased after 24 h at +4°C, the α₂-globulin percentage increased after 48 h at +4°C and at −20°C, and the γ-globulin percentage increased after 48 h at −20°C. The results should help veterinary practitioners handle and store equine serum samples appropriately. Further investigations at different storage times and temperatures could be useful.     

The challenges posed by equine arboviruses

Equine populations worldwide are at increasing risk of infection by viruses transmitted by biting arthropods, including mosquitoes, biting midges (Culicoides), sandflies and ticks. These include the flaviviruses (Japanese encephalitis, West Nile and Murray Valley encephalitis), alphaviruses (eastern, western and Venezuelan encephalitis) and the orbiviruses (African horse sickness and equine encephalosis). This review provides an overview of the challenges faced in the surveillance, prevention and control of the major equine arboviruses, particularly in the context of these viruses emerging in new regions of the world.     

Enhanced inactivation of avian influenza virus at ?20°C by disinfectants supplemented with calcium chloride or other antifreeze agents

Avian influenza outbreaks have occurred during winter months, and effective disinfection of poultry premises at freezing temperatures is needed. The commercial disinfectants Virkon and Accel, supplemented with an antifreeze agent [propylene glycol (PG), methanol (MeOH), or calcium chloride (CaCl₂)], were evaluated for their effectiveness in killing avian influenza virus (AIV) at −20°C or 21°C. An AIV suspension was applied to stainless steel disks, air-dried, and covered with a disinfectant or antifreeze agent for 5 to 30 min. Virkon (2%) and Accel (6.25%) with 30% PG, 20% MeOH, or 20% CaCl₂ inactivated 6 log₁₀ AIV within 5 min at −20°C and 21°C. At these temperatures PG and MeOH alone did not kill AIV, but the 20% CaCl₂ solution alone inactivated 5 log₁₀ AIV within 10 min. The results suggested that CaCl₂ is potentially useful to enhance the effectiveness of disinfection of poultry facilities after outbreaks of AIV infection in warm and cold seasons.     

Physical, Chemical and Biological Characteristics of Two Bovine Enterovirus Plaque Variants

Large plaque (4LP) and small plaque (4SP) variants were derived from a parent bovine virus strain by serial plaque passage. Both 4LP and 4SP were resistant to chloroform and stabilized at 50°C for one hour by 1.0 M magnesium chloride. Both 4LP and 4SP had buoyant densities in cesium chloride of 1.36 gm/ml. Antigenically, 4LP and 4SP were reciprocally cross neutralizable.

The nucleic acid of 4LP was shown to be ribonucleic acid (RNA) by resistance of its infectivity to deoxynuclease (DNase) but not ribonuclease (RNase) and by increased incorporation of [³H]-uridine into cytoplasmic RNA in cells of virus infected cultures. In growth characteristics, both 4LP and 4SP had maximum adsorption times of 75 to 90 minutes but 4LP had more rapid replication and release rates and yielded nearly twice as many infectious units per cell as 4SP. The differences in growth properties correlated directly with the differential in plaque diameter which was 40-50%.

     

Seroprevalence of equine granulocytic anaplasmosis and lyme borreliosis in Canada as determined by a point-of-care enzyme-linked immunosorbent assay (ELISA)

Equine granulocytic anaplasmosis (EGA) and Lyme borreliosis (LB) are an emerging concern in Canada. We estimated the seroprevalence of EGA and equine LB by testing 376 convenience serum samples from 3 provinces using a point-of-care SNAP^® 4Dx^® ELISA (IDEXX Laboratories, Westbrook, Maine, USA), and investigated the agreement between the point-of-care ELISA and laboratory-based serologic tests. The estimated seroprevalence for EGA was 0.53% overall (0.49% in Saskatchewan, 0.71% in Manitoba), while the estimated seroprevalence for LB was 1.6% overall (0.49% in Saskatchewan, 2.86% in Manitoba). There was limited agreement between the point-of-care ELISA and an indirect fluorescent antibody test for EGA (kappa 0.1, PABAK 0.47) and an ELISA/Western blot combination for LB (kappa 0.23, PABAK 0.71). While the SNAP^® 4Dx^® ELISA yielded expected seroprevalence estimates, further evaluation of serologic tests for the purposes of disease exposure recognition may be needed.     

The relative plasma availabilities of ivermectin in reindeer (Rangifer tarandus tarandus) following subcutaneous and two different oral formulation applications

Background

Overwintering (breeding) reindeer (Rangifer tarandus tarandus) are commonly treated with ivermectin against parasitic infestations once yearly in autumn-winter roundups. The only preparations registered to reindeer are those for subcutaneous injection. However, also oral extra-label ivermectin administration is used. Twenty-six, 8-month-old reindeer calves were randomly allocated into three groups. Group 1 (n = 9) received oral ivermectin mixture (Ivomec® vet mixt. 0.8 mg/ml, oral ovine liquid drench formulation), Group 2 (n = 9) oral ivermectin paste (Ivomec® vet 18.7 mg/g equine paste), and Group 3 (n = 8) subcutaneous injection of ivermectin (Ivomec® 10 mg/ml vet inj.), each group at a dose of 200 μg/kg body weight. Blood samples were collected at treatment and at days 1, 2, 3, 6, 9 and 16 post treatment. Plasma concentrations of ivermectin were determined by high-pressure liquid chromatography (HPLC) with fluorescence detection.

Results

The peak plasma concentration (C_max) was reached by 2 days after each treatment. The C_max and Area Under Curve (AUC) differed significantly between the groups: C_max was 30.2 ± 3.9, 14.9 ± 5.7 and 63.1 ± 13.1 ng/ml, and AUC_∞ was 2881 ± 462, 1299 ± 342 and 6718 ± 1620 ng*h/ml for groups 1, 2 and 3, respectively (mean ± standard deviation).

Conclusions

The differences in plasma concentrations of ivermectin are concomitant with earlier observed differences in antiparasitic efficacy, which discounts the use of the equine paste in reindeer in favour of the oral ovine liquid drench formulation, or preferably, the reindeer-registered subcutaneous injection formulation.     

Trypanosomes in Cattle in Southern Ontario

Trypanosomes were found in the blood of 155 out of 274 Holstein cows of various ages from southern Ontario. The incidence varied from about 8% in one herd to about 85% in another herd. Two species of trypanosomes were found in cattle in the Guelph area. One was Trypanosoma theileri. The other was a much smaller trypanosome which morphologically resembles T. uniforme of the Salivaria. This is the first report of such a trypanosome in cattle in North America. Epimastigotes from blood agar slants of both trypanosomes, when inoculated into chick embryos, produced blood forms morphologically identical to those found in the blood of the cows. T. theileri survived at least two weeks in blood kept at 4-5° C.     

Serological evidence of widespread exposure of Grenada fruit bats to chikungunya virus

Antibody detection against selected potentially zoonotic vector‐borne alphaviruses and flaviviruses was conducted on sera from bats from all six parishes in Grenada, West Indies. Sera were tested for (i) antibodies to flaviviruses West Nile virus, St. Louis encephalitis virus, Ilhéus virus, Bussuquara virus (BSQV), Rio Bravo virus and all four serotypes of dengue virus (DENV) by plaque reduction neutralization test (PRNT); (ii) antibodies to alphaviruses western equine encephalitis virus, Venezuelan equine encephalitis virus and eastern equine encephalitis virus by epitope‐blocking enzyme‐linked immunosorbent assay (ELISA); and (iii) antibodies to the alphavirus chikungunya (CHIKV) by PRNT. Two species of fruit bats were sampled, Artibeus jamaicensis and Artibeus lituratus, all roosting in or within 1,000 m of human settlements. Fifteen (36%) of the 42 bats tested for neutralizing antibodies to CHIKV were positive. The CHIKV‐seropositive bats lived in localities spanning five of the six parishes. All 43 bats tested for epitope‐blocking ELISA antibody to the other alphaviruses were negative, except one positive for Venezuelan equine encephalitis virus. All 50 bats tested for neutralizing antibody to flaviviruses were negative, except one that had a BSQV PRNT₈₀ titre of 20. The CHIKV serology results indicate that bats living close to and within human settlements were exposed to CHIKV in multiple locations. Importantly, bats for this study were trapped a year after the introduction and peak of the human CHIKV epidemic in Grenada. Thus, our data indicate that bats were exposed to CHIKV possibly during a time of marked decline in human cases.     

51-Chromium Labeled Erythrocyte Half-time Disappearance from Adult Sinclair (S-1) Miniature Swine

Half-time disappearance of ⁵¹Cr-labeled erythrocytes from 20 adult miniature swine was measured. Seven males and 13 females from 388 to 418 days of age, having a mean weight of 55kg were studied. Five ml of blood collected from each animal were labeled with 75 µCi ⁵¹Cr at room temperature for 60 to 90 min. Following injection of the labeled erythrocytes into each animal, blood samples were collected every four to six days. Specific activity was measured and half-time disappearance determined by linear regression. The results were compared with values obtained from the same animals tested at two earlier ages. No significant (P <0.05) difference in erythrocyte half-time disappearance was detected between males and females at the three different ages. Mean (±SEM) erythrocyte half-time disappearance values were: 20 to 46 days of age, 19.58 (±0.59) days; 79 to 109 days of age, 23.56 (±1.18) days; and 388 to 418 days of age, 22.35 (±0.72) days. A significant (P <0.01) difference in erythrocyte half-time disappearance was found between the miniature swine tested at 20 to 46 days of age and those tested at either 79 to 109 or 388 to 418 days of age. No significant (P <0.05) difference in erythrocyte half-time disappearance was found between the two older age groups.     

Incident Light Immunofluorescence of Alcohol-fixed Colonies of Ruminant Mycoplasma

Two species of ruminant mycoplasma colonies had to be fixed in ethyl alcohol so that incident immunofluorescence method could be applied. In addition, the stain reaction had to be kept for 90 minutes at 37°C.

This fluorescent antibody (FA) method was developed to identify colonies of Vom strain of Mycoplasma mycoides var. capri, V-5 strain of M. mycoides var. mycoides, and PG-2 strain of M. agalactiaeon agar, using fluorescent ultraviolet light. Fluorescence was not demonstrated when heterologous conjugates or normal rabbit serum conjugate were applied but the reaction appeared to be specific for each strain of mycoplasma.

The FA method was able to differentiate specific mycloplasma colonies in mixed cultures.

     

Two outer membrane proteins are bovine lactoferrin-binding proteins in Mannheimia haemolytica A1

Mannheimia haemolytica is a Gram negative bacterium that is part of the bovine respiratory disease, which causes important economic losses in the livestock industry. In the present work, the interaction between M. haemolytica A1 and bovine lactoferrin (BLf) was studied. This iron-chelating glycoprotein is part of the mammalian innate-immune system and is present in milk and mucosal secretions; Lf is also contained in neutrophils secondary granules, which release this glycoprotein at infection sites. It was evidenced that M. haemolytica was not able to use iron-charged BLf (BholoLf) as a sole iron source; nevertheless, iron-lacked BLf (BapoLf) showed a bactericidal effect against M. haemolytica with MIC of 4.88 ± 1.88 and 7.31 ± 1.62 μM for M. haemolytica strain F (field isolate) and M. haemolytica strain R (reference strain), respectively. Through overlay assays and 2-D electrophoresis, two OMP of 32.9 and 34.2 kDa with estimated IP of 8.18 and 9.35, respectively, were observed to bind both BapoLf and BholoLf; these OMP were identified by Maldi-Tof as OmpA (heat-modifiable OMP) and a membrane protein (porin). These M. haemolytica BLf binding proteins could be interacting in vivo with both forms of BLf depending on the iron state of the bovine.

Electronic supplementary material

The online version of this article (doi:10.1186/s13567-016-0378-1) contains supplementary material, which is available to authorized users.     

Seroepidemiology of Selected Alphaviruses and Flaviviruses in Bats in Trinidad

A serosurvey of antibodies against selected flaviviruses and alphaviruses in 384 bats (representing 10 genera and 14 species) was conducted in the Caribbean island of Trinidad. Sera were analysed using epitope‐blocking enzyme‐linked immunosorbent assays (ELISAs) specific for antibodies against West Nile virus (WNV), Venezuelan equine encephalitis virus (VEEV) and eastern equine encephalitis virus (EEEV), all of which are zoonotic viruses of public health significance in the region. Overall, the ELISAs resulted in the detection of VEEV‐specific antibodies in 11 (2.9%) of 384 bats. Antibodies to WNV and EEEV were not detected in any sera. Of the 384 sera, 308 were also screened using hemagglutination inhibition assay (HIA) for antibodies to the aforementioned viruses as well as St. Louis encephalitis virus (SLEV; which also causes epidemic disease in humans), Rio Bravo virus (RBV), Tamana bat virus (TABV) and western equine encephalitis virus (WEEV). Using this approach, antibodies to TABV and RBV were detected in 47 (15.3%) and 3 (1.0%) bats, respectively. HIA results also suggest the presence of antibodies to an undetermined flavivirus(es) in 8 (2.6%) bats. Seropositivity for TABV was significantly (P < 0.05; χ²) associated with bat species, location and feeding preference, and for VEEV with roost type and location. Differences in prevalence rates between urban and rural locations were statistically significant (P < 0.05; χ²) for TABV only. None of the aforementioned factors was significantly associated with RBV seropositivity rates.     

Detection of Antibodies to Alphaviruses and Discrimination between Antibodies to Eastern and Western Equine Encephalitis Viruses in Rabbit Sera using a Recombinant Antigen and Virus‐specific Monoclonal Antibodies

Three arthropod‐borne alphaviruses, western equine encephalitis viruses (WEEV), eastern equine encephalitis viruses (EEEV) and Venezuelan equine encephalitis viruses are the aetiological agents of a sometimes severe encephalomyelitis in equines and humans in the New World. With regard to the different ecology and epidemiology of these viruses, a method applied in serological screening should be able to distinguish between them as well as other related members of the genus Alphavirus in the American continent. However, this has been hampered in the past by (a) the close antigenic relationship between alphaviruses in traditional serological assays, especially in the routinely used haemagglutination‐inhibition, and (b) the need of biosafety level 3 facilities to grow the viral antigens. An epitope blocking assay using an EEEV glycoprotein E1‐expressing recombinant Sindbis virus and virus‐specific monoclonal antibodies (mAbs) binding to the E1 of EEEV (strain NJ/60) and the E1 of Sindbis virus was established using automated flow cytometry. The test was evaluated using sera of infected and vaccinated rabbits. A cut‐off value of 30% inhibition for antigenic complex‐specific seroconversion was found to be sufficient for the detection of the respective infection. By using three different mAbs in parallel, we were able to detect alphavirus genus‐, EEEV‐ and WEEV‐complex‐specific serum antibodies. As this test is based on the inhibition of binding of virus‐specific mAbs, sera of every origin other than mouse can be tested. Thus, this assay may prove useful in the serological screening of a variety of animal species during an outbreak investigation.     

Preanalytical variables affecting the measurement of serum paraoxonase-1 activity in horses

Paraoxonase-1 (PON-1) activity is a new inflammatory and oxidative marker. Technical effects and biological factors could affect the accuracy of PON-1 activity measurement. We investigated the effects of storage at different temperatures, repeated freeze–thaw cycles, interferences from hemolytic, lipemic, and icteric samples, and seasonal effects on PON-1 activity in horses. We evaluated 2 substrates with an automated spectrophotometer. Ten equine serum samples were stored under different conditions. Although storage at room (21°C) or refrigeration (4°C) temperature induced a statistically significant decrease (p < 0.05) in PON-1 activity, this is not diagnostically relevant. PON-1 activity in frozen samples (−20°C) was stable for short-term storage; diagnostically significant (p < 0.01) fluctuations were observed after 1 mo. Four repeated freeze–thaw cycles were assessed, and all cycles affected PON-1 activity (p < 0.01); however, this was diagnostically significant only after the 4th cycle. Hemolysis induced an overestimation of PON-1 activity; lipemia and hyperbilirubinemia did not change PON-1 activity. Thirty-four horses were sampled monthly for 1 y, and PON-1 activity was higher in autumn (p < 0.05) and winter (p < 0.05) than in spring and summer.     

Thermoregulatory and physiological responses of nonpregnant,mid-gestation,and late-gestation sows exposed to incrementally increasing dry bulb temperature

Gestating sows may be more susceptible to increasing dry bulb temperatures (T_DB) due to greater metabolic heat production and increased body mass, especially as gestation advances. However, there are few studies on the thermoregulatory and physiological responses of sows at differing gestation stages exposed to gradually increasing temperatures. The study objective was to determine the thermoregulatory and physiological responses of nonpregnant (n = 12; parity 3.27 ± 0.86), mid-gestation (59.7 ± 9.6 d pregnant, n = 12; parity 3.25 ± 0.83), and late-gestation (99.0 ± 4.8 d pregnant, n = 12; parity 3.33 ± 0.75) sows exposed to increasing T_DB. Prior to the experiment (5.0 ± 0.7 d), jugular catheters were placed in all sows. During the experiment, the T_DB was increased incrementally by 2.45 ± 0.43 °C every 60 min from 19.84 ± 2.15 to 35.54 ± 0.43 °C over 400 min, and relative humidity was recorded at 40.49 ± 18.57%. Respiration rate (RR), heart rate (HR), skin temperature, and vaginal temperature were measured, and blood samples were obtained via the jugular catheter every 20 min. Data were analyzed using PROC MIXED in SAS 9.4. RR increased at a lower T_DB (P < 0.01) in late-gestation sows compared with mid-gestation and nonpregnant sows, but no differences were detected between mid-gestation and nonpregnant sows. Overall, late-gestation sows had greater RR (P < 0.01; 23 ± 2 breaths per min [brpm]) compared with mid-gestation (16 ± 2 brpm) and nonpregnant (15 ± 2 brpm) sows. Late-gestation sows had an overall greater HR (P < 0.01; 84 ± 5 beats per min [bpm]) than mid-gestation (76 ± 5 bpm) and nonpregnant (69 ± 5 bpm) sows. Late-gestation sows had overall reduced bicarbonate and total carbon dioxide levels (P = 0.02; 23.89 ± 1.97 and 25.41 ± 2.07 mmol/L, respectively) compared with mid-gestation (27.03 ± 1.97 and 28.58 ± 2.07 mmol/L, respectively) and nonpregnant (26.08 ± 1.97 and 27.58 ± 2.07 mmol/L, respectively) sows. Moreover, late-gestation sows had overall greater nitric oxide levels (P < 0.01; 248.82 ± 34.54 µM) compared with mid-gestation (110.47 ± 34.54 µM) and nonpregnant (41.55 ± 34.54 µM) sows. In summary, late-gestation sows appear to be more sensitive to increasing T_DB as indicated by thermoregulatory and physiological responses when compared with mid-gestation or nonpregnant sows. The results from this study provide valuable information regarding thermoregulatory thresholds of sows at differing gestation stages.     

Safety and immunogenicity of a delta inulin-adjuvanted inactivated Japanese encephalitis virus vaccine in pregnant mares and foals

In 2011, following severe flooding in Eastern Australia, an unprecedented epidemic of equine encephalitis occurred in South-Eastern Australia, caused by Murray Valley encephalitis virus (MVEV) and a new variant strain of Kunjin virus, a subtype of West Nile virus (WNV_KUN). This prompted us to assess whether a delta inulin-adjuvanted, inactivated cell culture-derived Japanese encephalitis virus (JEV) vaccine (JE-ADVAX™) could be used in horses, including pregnant mares and foals, to not only induce immunity to JEV, but also elicit cross-protective antibodies against MVEV and WNV_KUN. Foals, 74–152 days old, received two injections of JE-ADVAX™. The vaccine was safe and well-tolerated and induced a strong JEV-neutralizing antibody response in all foals. MVEV and WNV_KUN antibody cross-reactivity was seen in 33% and 42% of the immunized foals, respectively. JE-ADVAX™ was also safe and well-tolerated in pregnant mares and induced high JEV-neutralizing titers. The neutralizing activity was passively transferred to their foals via colostrum. Foals that acquired passive immunity to JEV via maternal antibodies then were immunized with JE-ADVAX™ at 36–83 days of age, showed evidence of maternal antibody interference with low peak antibody titers post-immunization when compared to immunized foals of JEV-naïve dams. Nevertheless, when given a single JE-ADVAX™ booster immunization as yearlings, these animals developed a rapid and robust JEV-neutralizing antibody response, indicating that they were successfully primed to JEV when immunized as foals, despite the presence of maternal antibodies. Overall, JE-ADVAX™ appears safe and well-tolerated in pregnant mares and young foals and induces protective levels of JEV neutralizing antibodies with partial cross-neutralization of MVEV and WNV_KUN.